Journal of Medicinal Plants Research Vol. 5(17), pp. 4332-4338, 9 September, 2011 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875 ©2011 Academic Journals Full Length Research Paper Antibacterial activity of moroccan plants extracts against Clavibacter michiganensis subsp . michiganensis, the causal agent of tomatoes’ bacterial canker Talibi I., Amkraz N.*, Askarne L., Msanda F., Saadi B., Boudyach E. H., Boubaker H., Bouizgarne B. and Ait Ben Aoumar A. Faculté des Sciences, LBVRN ; Laboratoire de Biotechnologie et Valorisation des Ressources Naturelles, BP 8106, cité Dakhla, Agadir, 80 000, Morocco. Accepted 23 June, 2011 In search for alternative ways of tomatoes’ bacterial canker control, we screened here forty medicinal and aromatic plants (MAP) sampled from 15 families, currently used in southern Moroccan traditional medicine, for their activity against Clavibacter michiganensis subsp . michiganensis the causal agent of this disease. The antibacterial efficacy of powders of these plants was determined in vitro using the agar plate’s methods. Results obtained show that all the forty plants tested inhibited the bacterial growth of this pathogen with inhibition zone diameter ranging from 5 to 50 mm. Determination of the minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of the most effective plants indicates that there plants gender Rubus, Anvillea and Pistacia have the lower MIC which is equal to 3,125 mg ml -1. The other plants ( Lavandula coronopifolia, Lavandula stoechas, Rosa canina, Cistus monspliensis and Cistus crispus ) had a MIC equal to 6.25 mg ml -1. The MBC for different plants tested are between 6.25 mg ml -1, case of Rubus ulmifolius and 25 mg ml -1. The germination percentage of seeds treated with the most efficient three plants extracts was significantly higher than the untreated. Seeds treated with the extract of L. coronopifolia gave the higher germination rate (98%). The rate germination is 84 to 88% respectively for Cistus monspeliensis and Rubus ulmifolius . In other hand population size of the bacterium in the seeds’ surface was significantly reduced compared to the controls for the three treatments, when tomato seeds were artificially inoculated with the pathogen before being treated with the plants extracts (aqueous extracts). Key words: Bacterial canker, tomato, medicinal plants, biological control. INTRODUCTION The tomato crop is subject to attack by a multitude of and destructive disease in tomato crop both under pathogenic microorganisms and its intensive culture has controlled and field cultures (Utkhede and Koch, 2004). generated and amplified the phytosanitary problems. In The bacterial canker can cause significant damage which addition to mushrooms, viruses and deleterious may go up to the destruction of 100% crop (Gitaitis, 1990; organisms, pathogenic bacteria are important factors that Chang et al., 1992). In Morocco, all tomato production reduce the quality and performance of this culture areas are contaminated by this pathogen, whose (Gartemann et al., 2003). The bacterial canker caused by seriousness varies following regions (Fatmi et al., 1989). Clavibacter michiganensis subsp . michiganensis (Smith) In the Souss-Massa Draa region, it has become the main (Davis et al., 1984), account among the most important cause of the premature death of tomato (Fatmi, 1989). phytosanitary tomato problems. It is a very contagious The seeds and infected transplants are the primary sources of primary inoculums of C. michiganensis subsp . michiganensis (Bryan, 1930; Gleason et al., 1991). Bacteria can survive in the soil (Bryan, 1930; Chang et *Corresponding author. E-mail: [email protected]. al ., 1992; Strider, 1967; Farley, 1971), in crop residues Talibi et al. 4333 and multiple alternative hosts (Fatmi and Schaad, 2002). culture of 72 h on the NBY medium. The optical density of bacterial 8 -1 The spread of the disease is ensured by various suspension was adjusted to 0.46 at 610 nm to have 10 cfu ml manipulations like transplantation (Gitaitis, 1990; Gitaitis (Umesha, 2006). A volume of 0.1 ml of the suspension was speared in the medium surface with sterile glass beads. Cultures et al., 1991), phytosanitary treatments and irrigation prepared were subsequently placed in an incubator at 25°C for 30 systems (Strider, 1969; Chang et al., 1992). The min. Sterilized filter paper discs (Whatmann no 3 and 5 mm in chemical treatments recommended for this disease only diameter) were soaked with 10 µl of a 10% concentration of each reduced the population of the pathogen in the surface of plant aqueous extract. Discs were placed in the middle of Petri the infected plants (Hausbeck et al., 2000). Although the plates containing pathogen dried cultures prepared previously. Distilled water was used as negative controls. The plates were control of this bacterial disease continues to be difficult, incubated at 25°C and observed after 48 to 72 h. At the end of the prevention was the first defense line. Given the period, inhibition zones around the paper discs were measured and inefficiency of chemical treatments and their impact on compared to the controls. All tests were conducted in fifteen health and the environment, research and development repetitions arranged as five paper discs in three plates for each of alternative methods are recommended. The biocontrol extract. is a promising way and much research works were made worldwide against bacterial canker agent and various Determination of the MIC and the MBC of the efficient plants plant pathogens and encouraging results were fined (Amkraz et al., 2010; Taqarort et al., 2008; Ameziane et The minimal inhibitory concentrations (MIC) of aqueous plants al., 2007; Basim et al., 2006; Umesha, 2006; Utkhede extracts were determined in liquid medium (Schaad et al ., 1995). One suspension of C. michiganensis subsp . michiganensis (H195 and Koch, 2004; Dimitra et al., 2003; Boudyach et al., 8 -1 2004; Ali-Emmanuel et al., 2002; Boudyach et al., 2001). isolate) in exponential growth phase, adjusted to 10 cfu ml was distributed into sterile hemolytic tubes 3.6 ml per tube. A volume of With a view to develop effective natural treatments, 0.4 ml from the stock solution of the plant extract was added to natural extracted substances from plants figure as each tube. We performed a series of 1/2 dilution. The final privileged axes of the biological control of plant concentrations range between 50 and 3,125 mg l -1. Controls pathogens both in crop treatments and in post harvest consisted in tubes with the pathogen suspension without extract. manages. The objective of this study is to select among The tubes were then incubated at 25°C during 24 h. After incubation, the first tube showing visible growth to the naked eye plants harvested in different regions Morocco those which was retained as corresponding to the MIC for each plant. extracts inhibit the growth of C. michiganensis subsp . The minimal bactericidal concentration (MBC) was determined by michiganensis and to assess their activity in vitro. We are spreading 10 µl, taken from the MIC tubes, on the NBY agar. The also looking for a biological alternative to the chemical to percentage of viable germs was estimated comparing with controls eradicate the pathogen from the seeds surface performed with decimal dilutions of the bacterial suspension in considered to be the primary source of infection; in the order to obtain a range suspension of bacterial concentrations between 100 and 0.01%. Each dilution 10 µl was spread on NBY aim to have a certified seeds without C. michiganensis medium. Cultures thus made were incubated at 25°C for 72 h. The subsp . michiganensis. MBC that corresponds to the plant does allow extract concentration than 0.01% survival of the initial inoculums, was estimated by comparing the number of colonies appeared to that of the initial MATERIALS AND METHODS inoculums with the tenfold dilution series. The MIC of solvent extracts was determined during the previous assay; it corresponds Plant sampling and preparation of the plants extracts to the lower concentration that inhibited C. michiganensis subsp. michiganensis growth. The prospecting and collection of the medicinal plants were conducted during the years 2006, 2007 and 2008 from several regions of Souss-Massa Draa Valley, Agadir, Morocco. The plants Effectiveness of the aqueous extract in the treatment of the harvested were placed in clean plastic bags and numbered. After tomato infected seeds identification in the laboratory, the samples were dried in the shade and in damp shelter for a week, followed by a drying in an oven at The three best plants which extracts inhibit the growth of C. 35°C for one night. After drying, different parts of the plants were michiganensis subsp . michiganensis were used for treating infected transformed to powder. 20 g of powder were put into suspension in tomato seeds. The treatment of seeds and assessment of their one beaker 250 ml containing 60 ml of distilled water. All was germination capacity were made using the method of Tobias et al. brought to a boil during 5 min. Then, the obtained suspensions (2007) modified. Contaminated tomato seeds were mixed with the were double filtered utilizing the gauze followed by filter paper aqueous extract of each plant at a concentration of 500 mg ml -1 for (Whatmann no 1). The filtrate thus obtained was dried in an oven at one hour. Seeds were subsequently dried and then planted in 60°C to get dry extract. The stock solution of each extract alveolus terrines fulfilled with mixture of sand soil and peat (1: 2; corresponded to a concentration of 0.5 g dry extract in 1 ml of v/v). Each treatment was repeated 17 times and each test was distilled water (Ali-Emmanuel et al., 2002). repeated three times. Controls were carried out under the same conditions with seeds dipped in distilled water. The terrines were placed in an experimental greenhouse with randomized design Screening for the antibacterial activity of the aqueous extracts blocs and were watered once every two days.